Methods for preventing progressive tissue necrosis, reperfusion injury, bacterial translocation and adult respiratory distress syndrome

ABSTRACT

The present invention is directed to a method for preventing or reducing ischemia following injury, such as reperfusion injury following ischemia, cellular damage associated with ischemic episodes, such as infarctions or traumatic injuries, and thus to prevent or reduce the consequent progressive necrosis of tissue associated with such ischemia. This effect is achieved by administering DHEA, DHEA derivatives or DHEA congeners to a patient as soon as possible after the injury. The present invention is further directed to methods for preventing or reducing bacterial translocation or adult respiratory distress syndrome in a patient. Similarly, bacterial translocation and adult respiratory distress syndrome are prevented or reduced by administering DHEA, DHEA derivatives or DHEA congeners to a patient.

This invention was made with Government support under GrantN00014-92-J-1612 awarded by the Department of the Navy. The Governmenthas certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part application ofapplication Ser. No. 08/284,688, now U.S. Pat. No. 5,532,230 filed 9Aug. 1994, which is a continuation-in-part applicaiton of applicationSer. No. 08/029,422, filed 9 Mar. 1993; now abandoned the specificationof each is incorporated herein.

BACKGROUND OF THE INVENTION

The present invention is related to a method for preventing or reducingthe effects of ischemia. The ischemia may be associated with injury,such as occurs as a result of infarctions, thermal injury (burns),surgical trauma, accidental trauma and the like. The ischemia may alsoprecede reperfusion injury. The invention is also related to methods forpreventing or reducing bacterial translocation and adult respiratorydistress syndrome. In accordance with the present invention, theseconditions are prevented by administering dehydroepiandrosterone (DHEA)or DHEA derivatives.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular cases, to provideadditional details respecting the practice, are incorporated byreference, and for convenience are numerically referenced in thefollowing text and respectively grouped in the appended bibliography.

It has been recognized that the maintenance of vascular integrity is animportant response to injury. Complex hemostatic mechanisms ofcoagulation, platelet function and fibrinolysis exist to minimizeadverse consequences of vascular injury and to accelerate vascularrepair. Vascular endothelial and smooth muscle cells actively maintainvessel wall thromboresistance by expressing several antithromboticproperties. When perturbed or injured, vascular cells expressthrombogenic properties. The hemostatic properties of normal andperturbed vascular cells has been reviewed by Rodgers (1).

Interference with the supply of oxygenated blood to tissues is definedas ischemia. The effects of ischemia are known to be progressive, suchthat over time cellular vitality continues to deteriorate and tissuesbecome necrotic. Total persistent ischemia, with limited oxygenperfusion of tissues, results in cell death and eventually incoagulation-induced necrosis despite reperfusion with arterial blood.Ischemia is probably the most important cause of coagulative necrosis inhuman disease. A substantial body of evidence claims that a significantproportion of the injury associated with ischemia is a consequence ofthe events associated with reperfusion of ischemic tissues, hence theterm reperfusion injury. To place reperfusion injury into a clinicalperspective, there are three different degrees of cell injury, dependingon the duration of ischemia:

(1) With short periods of ischemia, reperfusion (and resupply of oxygen)completely restores the structural and functional integrity of the cell.Whatever degree of injury the cells have incurred can be completelyreversed upon reoxygenation. For example, changes in cellular membranepotential, metabolism and ultrastructure are short-lived if thecirculation is rapidly restored.

(2) With longer periods of ischemia, reperfusion is not associated withthe restoration of cell structure and function, but rather withdeterioration and death of cells. The response to reoxygenation in thiscase is rapid and intense inflammation.

(3) Lethal cell injury may develop during prolonged periods of ischemia,where reperfusion is not a factor.

The reversibility of cell injury as a consequence of ischemia isdetermined not only by the type and duration of the injury, but also bythe cell target. Neurons exhibit very high sensitivity to ischemia,whereas myocardial, pulmonary, hepatic and renal tissues areintermediate in sensitivity. Fibroblasts, epidermis and skeletal musclehave the lowest susceptibility to ischemic injury, requiring severalhours without blood supply to develop irreversible damage.

The proximity of the endothelium to circulating leukocytes makes it animportant early target for neutrophil adherence and subsequent damage tovascular and parenchymal tissue. Interaction of activated endothelialcells and neutrophils is an immediate early, and necessary, event inischemia/reperfusion injury (2, 3). The adhesive properties ofendothelium are rapidly induced by the influx of oxygenated blood. Inresponse to oxygen, endothelial cells become activated to produceseveral products, including leukotriene B4 (LTB4), platelet activatingfactor (PAF) and P-selectin. Leukotriene B4 is a potent neutrophilchemotactic agent (4, 5). Upon activation of the endothelial cells,P-selectin is rapidly translocated from intracellular organelles to theplasma membrane, where it acts to tether circulating neutrophils andstabilize them for activation by endothelial-bound PAF (plateletactivating factor), enddothelium-derived cytokines and otherbiologically active mediators (6). Thus, the physiologic interactionbetween the activated endothelium and the activated neutrophil isrecognized as a critical and immediate early event in reperfusion injuryof organs and tissues. Other cellular and biochemical mediators ofinflammation injury such as platelets, the complement cascade, and thecoagulation system are also important, but come into play much later inthe cascade, in a process called coagulative necrosis. Finally,monocytes, macrophages, fibroblasts and smooth muscle cell infiltrationare responsible for reconstruction and replacement of dead tissue withnew, vital tissue, a process called wound healing.

A popular theory postulates a role for partially reduced, and thusactivated, oxygen species in the initiation of membrane damage inreperfusion injury. Present evidence indicates that activated oxygen(superoxide, peroxide, hydroxyl radicals) is formed during ischemicepisodes and that reactive oxygen species injure ischemic cells. Toxicoxygen species are generated not during the period of ischemia itself,but rather on restoration of blood flow, or reperfusion. Two sources ofactivated oxygen species have been implicated as early events inreperfusion injury, those produced intracellularly by the xanthineoxidase pathway and those which can be transported to the extracellularenvironment by activated neutrophils (2, 3, 7-9).

In the xanthine oxidase-dependent pathway, purines derived from thecatabolism of ATP during the ischemic period provide substrates for theactivity of xanthine oxidase, which requires oxygen in catalyzing theformation of uric acid. Activated oxygen species are byproducts of thisreaction. The species of oxygen radicals derived from the xanthineoxidase pathway are O₂ ⁻ (superoxide with one electron) and H₂ O₂(hydrogen peroxide with two unpaired electrons). Superoxides aregenerated within the cytosol by xanthine oxidase (located in thecytosol). The superoxides are then catabolized to peroxides withinmitochondria by superoxide dismutase. The peroxides are furtherconverted to water either by glutathione peroxidase, in the cytosol, orby catalase in peroxisomes. Both glutathione peroxidase and catalasecomprise the antioxidant defense mechanism of most cells. The majorevidence for this hypothesis rests on the ability of allopurinol, aninhibitor of xanthine oxidase, to protect against reperfusion injury inexperimental models.

In the NADPH-dependent pathway, NADPH oxidase is activated to generatesuperoxides through reduction of molecular oxygen at the plasmamembrane. The superoxides are reduced to hydrogen peroxide by superoxidedismutase at the plasma membrane or within phagolysosomes. Finally,hydrogen peroxide within phagolysosomes can be reduced in the presenceof superoxides or ferrous iron to hydroxyl radicals. A third form ofoxygen metabolite is mediated by myloperoxidase in the presence ofchlorine to reduce hydrogen peroxide to hypochlorous acid.

The hydroxyl radical is an extremely reactive species. Mitochondrialmembranes offer a number of suitable substrates for attack by OH⁻radicals. The end result is irreversible damage to mitochondria,perpetuated by a massive influx of Ca²⁺ ions. Another probable cause ofcell death by hydroxyl radicals is through peroxidation of phospholipidsin the plasma membrane. Unsaturated fatty acids are highly susceptibletargets of hydroxyl radicals. By removing a hydrogen atom from fattyacids of cell membrane phospholipids, a free lipid radical is formed.These lipid radicals function like hydroxyl radicals to form other lipidperoxide radicals. The destruction of unsaturated fatty acids ofphospholipids leads to a loss in membrane fluidity and cell death. Someinvestigators believe that the effects of oxidative stress causeprogrammed cell death in a variety of cell types.

Infarctions and traumatic injury involve many tissues, includingvascular tissue. One response following traumatic injury is to shut downblood supply to the injured tissue. A purpose of this response is toprotect the patient from the entry of infectious agents into the body.The severe reduction in blood supply is a main factor leading toprogressive ischemia at the region of the traumatic injury. Withprogressive ischemia, tissue necrosis extends beyond the directlyaffected tissue to include surrounding unaffected tissue. Thisprogressive ischemia plays an important role in defining the ultimatetissue pathology observed in humans as a consequence of the traumaticinjury. For example, see Robson et al. (10).

One form of traumatic injury which has received a great deal ofattention is thermal injury or burns. The burn wound represents anon-uniform injury, and the spectrum of injury ranges from tissue whichis totally coagulated at the time of injury to tissue which is onlyminimally injured. Between these two extremes is tissue which isseriously damaged and not immediately destroyed, but which is destinedto die. The etiology of the progressive depth of necrosis has been shownto be stasis and thrombosis of blood flow in the dermal vessels, causingischemia and destruction of epithelial elements. This ischemia occursfor 24-48 hours following the thermal injury (10, 11). Many effects havebeen seen following a thermal injury, including adhesion of leukocytesto vessel walls, agglutination of red blood cells and liberation ofvasoactive and necrotizing substances (11).

It has been established that burn-associated micro-vascular occlusionand ischemia are caused by the time dependent increase in development ofmicrothrombi in the zone of stasis, a condition which eventually leadsto a total occlusion of the arterioles and a microcir-culatorystandstill. Whereas margination of erythrocytes, granulocytes andplatelets on venular walls are all apparent within the first few hoursfollowing thermal injury, the formation of platelet microthrombi(occurring approximately 24 hours after surgery) is believed to beresponsible for creating the conditions that cause complete andpermanent vascular occlusion and tissue destruction (12, 13). Theformation of platelet microthrombi appears to provide the cellular basisfor expanding the zone of complete occlusion and the ischemic necrosisthat advances into the zone of stasis following thermal injury.

Much effort has been made toward improving the care of burns and othertraumatic injuries, and many approaches have been proposed towardreducing the progressive ischemia associated with such injuries. Theanti-inflammatory agents indomethacin, acetylsalicylic acid andmethylprednisone acetate have been shown to preserve dermal perfusion(10). Three thromboxane inhibitors, imidazole, methimazole anddipyridamole, have been shown to prevent vascular changes in the burnwound, allow dermal perfusion and allow other prosta-glandin synthesis,which would circumvent detrimental effects of the anti-inflammatoryagents (11). Therapeutic doses of ibuprofen and imidazole were found toprevent dermal vascular occlusion by acting as an antagonist to aplasmin inhibitor (14). The reduction of circulating fibrinogen, shownby administration of ancrod (a pit viper venom), led to preservation ofvascular potency at the site of the injury (15). It has also been foundthat the inhibition of leukocyte-endothelial adherence, shown by usingmonoclonal anti-bodies, prevents burn extension/progression in themarginal zone of stasis (16).

Bacterial translocation is the process by which indigenous gut florapenetrate the intestinal barrier and invade sterile tissue. Included inthis process is the migration of microbial organisms to the drainingmesenteric lymph nodes, spleen, liver, blood and in some instances, thelung (17, 18). This phenomenon has been documented in humans followingthermal injury (19-21) and ischemia-reperfusion injury (22).

Under normal conditions, the intestinal mucosa is impermeable topotentially harmful materials from the intestinal lumen (17, 22, 23).Current data supports the concept that disruption of theintegrity/permeability of the mucosa promotes bacterial translocation,since exposure to stress which produces a host response characterized bycellular damage and necrotic tissue correlates with development ofbacterial translocation (23). The clinically important repercussions ofbacterial translocation are sepsis and multi-system organ failure(22-24). The incidence of sepsis and disseminated organ involvementfollowing stress is greatest among patients that also exhibitcompromised immune defenses (22, 23), such as observed in thermallyinjured individuals (24, 25). Thus, in response to stress, some patientsdemonstrate bacterial translo-cation in the absence of severeconsequences. The patients in this category are those who have retainedintact immune defenses (22-24). Because of the well known modulation ofthe host immune defenses following severe burn, bacterial translocationis one of the more serious consequences of thermal injury in humans (24,25).

Experimental models of bacterial translocation have noted thatirreversible cellular injury of the gut may require up to 24 hourspost-thermal injury and 48 hours to visualize histological changes ingut vascular tissue (21, 26). These experimental systems have beenuseful in defining the pharmacologic mediators which appear to formulatea cascade of effector molecules responsible for tissue necrosis. Inaddition to the role played by catecholamines, oxygen-free radicals andendotoxin, factors such as interferon alpha, interleu-kin-6, tumornecrosis factor, platelet activating factor, and many of the vasoactivefatty acids derived from arachidonic acid metabolism have beenimplicated (17). The contribution of oxygen-free radicals, endo-toxin,prostaglandins and thromboxanes in promoting tissue destruction has beensupported by the evidence that inhibition of bacterial translocation andmucosal injury has been achieved using allopurinol (27) (an inhibitor ofxanthine oxidase), endotoxin desensitiza-tion (28), prostaglandinanalogs (29) and thromboxane synthetase inhibitors (30).

The evidence implicating the role of neutrophils in adult respiratorydistress syndrome (ARDS) is substantial but indirect (31). Some of thefirst suggestions that neutrophils may cause an ARDS-like picture werefound in severely neutropenic patients who were infused intravenouslywith donor neutrophils. Occasionally, within hours of neutrophilinfusion, there was an abrupt "white-out" of the lungs Coy x-ray) andonset of ARDS symptoms. Numerous studies have shown that neutrophilsaccumulate in the lung during ARDS. For example, their presence has beendemonstrated histologically. During the early phases of ARDS, the numberof circulating whole blood cells transiently decreases, probably due totheir abnormal pulmonary sequestration. Some neutrophils that accumulatewithin lung capillaries leave the vascular space and migrate into theinterstitium and alveolar airspaces. In normal healthy volunteers,neutrophils account for less than 3% of the cells that can be obtainedby bronchoalveolar lavage (BAL). In patients with ARDS, the percentageof neutrophils in the lavage is markedly increased to 76-85%. Theaccumulation of neutrophils is associated with evidence of theiractivation. They demonstrate enhanced chemotaxis and generate abnormallyhigh levels of oxygen metabolites following in vitro stimulation.Elevated concentrations of neutrophil secretory products, such aslactoferrin, have been detected in the plasma of patients with ARDS.Further evidence that neutrophils actively participate in lung injurywas obtained from a clinical study of patients with mild lung injury whowere neutropenic for an unrelated reason (e.g., receiving chemotherapy).It was noted that lung impairment frequently worsened if a patient'shematological condition improved and circulating neutrophil countsrecovered to normal levels.

Although the evidence implicating neutrophils in the genesis of humanARDS is still largely indirect, data demonstrating the importance ofneutrophils in various animal models of acute lung injury is convincing.The common approach that has been used to demonstrate neutrophilindependence is to deplete the animal of circulating neutrophils andmeasure any diminution in lung injury that occurs. Although a number ofexperimental models have been used to study neutrophil dependence oflung injury, only a few have been selected for discussion herein becauseof space limitations.

One extensively studied model is the administration of endotoxin tosheep. When endotoxin is intravenously infused into sheep, a complex setof events occurs, one of which is increased permeability of thepulmonary capillary endothelium. This is manifested by an increase inthe flow of lung lymph which contains a higher-than-normal proteinconcentration. These changes indicate a reduction in the ability of thecapillary endothelium to retain plasma proteins within the vascularspace. The neutrophil dependence of the permeability injury wasestablished when it was found that neutrophil depletion of the sheepprior to endotoxin infusion protected them. Another in vitro model ofacute lung injury involves the intravenous infusion of cobra venomfactor into rats, which causes complement activation followed byleukoaggregation and sequestration of neutrophils within the pulmonarymicrovasculature. Alveolar wall damage occurs, leading to interstitialand intra-alveolar edema with hemorrhage and fibrin deposition. Again,neutrophil depletion prevented the increased pulmonary capillary leak.

Isolated, perfused rabbit or rat lungs have also been used to studymechanisms of alveolar injury under circumstances that allow improvedcontrol of the variables that affect fluid flux. When neutrophils wereadded to the perfusate and then stimulated, albumin leaked from thevascular compartment into the lung interstitium and alveolar airspaces.Unstimulated neutrophils or stimulus alone (e.g., phorbol myristateacetate) failed to increase alveolar-capillary permeability.

As further proof that stimulated neutrophils can independently injurelung tissue, in vitro experiments have been performed using vascularendothelial and lung epithelial cells as targets. In some reports,neutrophils have been shown to detach endothelial cells or alveolarepithelial cells from the surface of the tissue culture dish. Obviously,if such an event were to occur in vivo, the denuded surfaces wouldpermit substantial leakage of plasma contents. Furthermore, many reportshave provided clear evidence that stimulated neutrophils are able tofacilitate lysis l0 of cultured vascular endothelial cells and alveolarepithelial cells.

DHEA is an endogenous androgenic steroid which has been shown to have amyriad of biological activities. Araneo et al (32) has shown that theadministration of DHEA to burned mice within one hour after injuryresulted in the preservation of normal immunologic competence, includingthe normal capacity to produce T-cell-derived lymphokines, thegeneration of cellular immune responses and the ability to resist aninduced infection. Eich et al (33, 34) describes the use of DHEA toreduce the rate of platelet aggregation and the use of DHEA orDHEA-sulfate (DHEA-S) to reduce the production of thromboxane,respectively.

Nestler et al. (35) shows that administration of DHEA was able in humanpatients to reduce body fat mass, increase muscle mass, lower LDLcholesterol levels without affecting HDL cholesterol levels, lower serumapolipoprotein B levels, and not affect tissue sensitivity to insulin.Kent (36) reported DHEA to be a "miracle drug" which may preventobesity, aging, diabetes millitus and heart disease. DHEA was widelyprescribed as a drug treatment for many years. However, the Food andDrug Administration recently restricted its use. DHEA is readilyinterconvertible with its sulfate ester DHEA-S through the action ofintracellular sulfatases and sulfotransferases.

Despite the above discoveries concerning effects of various compounds onburns, there is a need to identify additional compounds which are ableto prevent or reduce reperfusion injury as a consequence of ischemia,effects of ischemia associated with infarctions or traumatic injury, andto identify compounds which are able to prevent or reduce bacterialtranslocation and ARDS. Thus, it is an object of the present inventionto prevent or reduce progressive tissue necrosis, to prevent or reducereperfusion injury, to prevent or reduce bacterial translocation, and toprevent or reduce ARDS.

SUMMARY OF THE INVENTION

The present invention is directed to a method for preventing or reducingreducing reperfusion injury following ischemia, cellular damageassociated with ischemic episodes, such as infarction or traumaticinjury, and thus to prevent or reduce the consequent progressivenecrosis of tissue associated with such ischemia. The present inventionis also directed to a method for preventing or reducing bacterialtranslocation. The present invention is further directed to a method forpreventing or reducint ARDS. Finally, the present invention is directedto a method for inhibiting the expression of p-selectin on endothelium.Reperfusion injury is prevented or reduced by administering DHEA, DHEAderivatives or DHEA congeners (collectively referred to as DHEAcongeners) to a patient following, e.g., an infarction or traumaticinjury. Similarly, bacterial translocation is prevented or reduced in apatient by administering DHEA congeners. ARDS is also prevented orreduced in a patient by administering DHEA congeners. Similarly,p-selectin expression by the endothelium is prevented or reduced in apatient by administering DHEA congeners.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of the analysis of edema formation (earswelling) and resolution in the burned ears of control and DHEA-treatedmice.

FIG. 2 shows the analysis of edema formation (ear swelling) andresolution in the burned ears of control mice and mice treated withDHEA, androstenediol, 16a-bromo-DHEA or the known anti-glucocorticoidRU486.

FIG. 3A shows the capacity of DHEA to protect against most of theprogressive ischemia consequences of thermal injury to the ear.

FIG. 3B shows the capacity of androstenediol to protect against most ofthe progressive ischemia consequences of thermal injury to the ear.

FIG. 3C shows the capacity of 16a-bromo-DHEA to protect against most ofthe progressive ischemia consequences of thermal injury to the ear.

FIG. 3D shows the progressive ischemic consequences of thermal injury tothe ear when vehicle alone is administered.

FIG. 3E shows the progressive ischemic consequences of thermal injury tothe ear when androstenedione alone is administered.

FIG. 3F shows the progressive ischemic consequences of thermal injury tothe ear when RU486 alone is administered.

FIG. 4 shows the effect of treatment with DHEA on progressive ischemiawhen administered from 0-6 hours post-thermal injury.

FIG. 5A shows the number of flowing capillaries in proximity topost-capillary venule in Zone 1 during reperfusion injury.

FIG. 5B shows the number of flowing capillaries in proximity topost-capillary venule in Zone 2 during reperfusion injury.

FIG. 5C shows the number of flowing capillaries in proximity topost-capillary venule in Zone 3 during reperfusion injury.

FIG. 6A shows the number of leukocytes rolling through the lumen ofpost-capillary venules in a two-minute period.

FIG. 6B shows the number of leukocytes adhering or sticking to the lumenof post-capillary venules in a two-minute period.

FIG. 6C shows the number of leukocytes migrating across the endotheliumin a two-minute period.

FIG. 7A shows red cell velocity of venous blood post-reperfusion.

FIG. 7B shows red cell velocity of arterial blood post-reperfusion.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for preventing or reducingreperfusion injury following ischemia, and cellular damage associatedwith ischemic episodes, such as infarction or traumatic injury. Anexample of an infarction is a myocardial infarction. Examples oftraumatic injury include thermal injury, surgery, chemical burns, blunttrauma or lacerations and the like. By preventing or reducingreperfusion injury following ischemia and cellular damage associatedwith ischemic episodes, the consequent progressive necrosis of tissueassociated with such infarction or injury is also prevented or reduced.In accordance with the present invention, reperfusion injury or cellulardamage associated with ischemic episodes, such as infarction ortraumatic injury, is prevented or reduced by administering DHEA, DHEAderivatives or DHEA congeners to a patient as early as possible,preferably within four hours, and most preferably within two hours, ofthe ischemia, infarction or traumatic injury.

The present invention is also directed to a method for preventing orreducing bacterial translocation. In accordance with the presentinvention, bacterial translocation is prevented or reduced in a patientby administering DHEA, DHEA derivatives or DHEA congeners as describedabove. The DHEA congeners is administered within 24 hours of an injuryin which bacterial translocation is one of the sequelae.

The present invention is also directed to a method for preventing orreducing adult respiratory distress syndrome (ARDS). In accordance withthe present invention, ARDS is prevented or reduced in a patient byadministering DHEA, DHEA derivatives or DHEA congeners as describedabove. The DHEA congeners is administered prior to clinical symptoms ofARDS, primarily to individuals at risk for ARDS.

Examples of "DHEA", "DHEA congener" or "DHEA-derivative", include butare not limited to, compounds having the formula ##STR1## wherein X is Hor halogen;

R¹, R² and R³ are independently ═O, --OH, --SH, H, halogen,pharmaceutically acceptable ester, pharmaceutically acceptablethioester, pharmaceutically acceptable ether, pharmaceuticallyacceptable thioether, pharmaceutically acceptable inorganic esters,pharmaceutically acceptable monosaccharide, disaccharide oroligosaccharide, spirooxirane, spirothirane, --OSO₂ R⁵ or --OPOR⁵ R⁶ ;

R⁵ and R⁶ are independently --OH, pharmaceutically acceptable esters orpharmaceutically acceptable ethers; and

pharmaceutically acceptable salts.

Thus, examples of suitable DHEA congeners include compounds in which:

(1) R² is ═O, R³ and X are each H and R¹ is ═O, --OH, pharmaceuticallyacceptable esters thereof, pharmaceutically acceptable ethers thereof orpharmaceutically acceptable salts;

(2) R² is ═O, R³ is H, X is halogen and R¹ is ═O, --OH, pharmaceuticallyacceptable esters thereof, pharmaceutically acceptable ethers thereof orpharmaceutically acceptable salts;

(3) R² is ═O, R³ and X are each H and R¹ is --SH, pharmaceuticallyacceptable thioesters thereof, pharmaceutically acceptable thioethersthereof or pharmaceutically acceptable salts;

(4) R² is ═O, R³ is H, X is halogen and R¹ is --SH, pharmaceuticallyacceptable thioesters thereof, pharmaceutically acceptable thioethersthereof or pharmaceutically acceptable salts;

(5) R² is ═O, X is H and R¹ and R³ are independently ═O, --OH,pharmaceutically acceptable esters thereof, pharmaceutically acceptableethers thereof or pharmaceutically acceptable salts;

(6) R² is ═O, X is halogen and R¹ and R³ are independently ═O, --OH,pharmaceutically acceptable esters thereof, pharmaceutically acceptableethers thereof or pharmaceutically acceptable salts;

(7) R² is ═O, X is H and R¹ and R³ are independently --SH,pharmaceutically acceptable thioesters thereof, pharmaceuticallyacceptable thioethers thereof or pharmaceutically acceptable salts;

(8) R² is ═O, X is halogen and R¹ and R³ are independently --SH,pharmaceutically acceptable thioesters thereof, pharmaceuticallyacceptable thioethers thereof or pharmaceutically acceptable salts;

(9) R² is --OH, R³ and X are each H and R¹ is ═O, --OH, pharmaceuticallyacceptable esters thereof, pharmaceutically acceptable ethers thereof orpharmaceutically acceptable salts;

(10) R² is --OH, R³ is H, X is halogen and R¹ is ═O, --OH,pharmaceutically acceptable esters thereof, pharmaceutically acceptableethers thereof or pharmaceutically acceptable salts;

(11) R² is --OH, R³ and X are each H and R¹ is --SH, pharmaceuticallyacceptable thioesters thereof, pharmaceutically acceptable thioethersthereof or pharmaceutically acceptable salts;

(12) R² is --OH, R³ is H, X is halogen and R¹ is --SH, pharmaceuticallyacceptable thioesters thereof, pharmaceutically acceptable thioethersthereof or pharmaceutically acceptable salts;

(13) R² is --OH, X is H and R¹ and R³ are independently ═O, --OH,pharmaceutically acceptable esters thereof, pharmaceutically acceptableethers thereof or pharmaceutically acceptable salts;

(14) R² is --OH, X is halogen and R¹ and R³ are independently ═O, --OH,pharmaceutically acceptable esters thereof, pharmaceutically acceptableethers thereof or pharmaceutically acceptable salts;

(15) R² is --OH, X is H and R¹ and R³ are independently --SH,pharmaceutically acceptable thioesters thereof, pharmaceuticallyacceptable thioethers thereof or pharmaceutically acceptable salts;

(16) R² is --OH, X is halogen and R¹ and R³ are independently --SH,pharmaceutically acceptable thioesters thereof, pharmaceuticallyacceptable thioethers thereof or pharmaceutically acceptable salts;

(17) R² is --SH, R³ and X are each H and R¹ is ═O, --OH,pharmaceutically acceptable esters thereof, pharmaceutically acceptableethers thereof or pharmaceutically acceptable salts;

(18) R² is --SH, R³ is H, X is halogen and R¹ is ═O, --OH,pharmaceutically acceptable esters thereof, pharmaceutically acceptableethers thereof or pharmaceutically acceptable salts;

(19) R² is --SH, R³ and X are each H and R¹ is --SH, pharmaceuticallyacceptable thioesters thereof, pharmaceutically acceptable thioethersthereof or pharmaceutically acceptable salts;

(20) R² is --SH, R³ is H, X is halogen and R¹ is --SH, pharmaceuticallyacceptable thioesters thereof, pharmaceutically acceptable thioethersthereof or pharmaceutically acceptable salts;

(21) R² is --SH, X is H and R¹ and R³ are independently ═O, --OH,pharmaceutically acceptable esters thereof, pharmaceutically acceptableethers thereof or pharmaceutically acceptable salts;

(22) R² is --SH, X is halogen and R¹ and R³ are independently ═O, --OH,pharmaceutically acceptable esters thereof, pharmaceutically acceptableethers thereof or pharmaceutically acceptable salts;

(23) R² is --SH, X is H and R¹ and R³ are independently --SH,pharmaceutically acceptable thioesters thereof, pharmaceuticallyacceptable thioethers thereof or pharmaceutically acceptable salts;

(24) R² is --SH, X is halogen and R¹ and R³ are independently --SH,pharmaceutically acceptable thioesters thereof, pharmaceuticallyacceptable thioethers thereof or pharmaceutically acceptable salts;

(25) X is H and R¹, R² and R³ are independently ═O, --OH, a sugarresidue, pharmaceutically acceptable esters thereof, pharmaceuticallyacceptable ethers thereof or pharmaceutically acceptable salts, whereinat least one of R¹, R² and R³ is a sugar residue;

(26) X is halogen and R¹, R² and R³ are independently ═O, --OH, a sugarresidue, pharmaceutically acceptable esters thereof, pharmaceuticallyacceptable ethers thereof or pharmaceutically acceptable salts, whereinat least one of R¹, R² and R³ is a sugar residue;

(27) X is H and R¹, R² and R³ are independently ═O, --OH,pharmaceutically acceptable inorganic esters thereof or pharmaceuticallyacceptable salts, wherein at least one of R¹, R² and R³ is an inorganicester;

(28) X is halogen and R¹, R² and R³ are independently ═O, --OH,pharmaceutically acceptable inorganic esters thereof or pharmaceuticallyacceptable salts, wherein at least one of R¹, R² and R³ is an inorganicester.

Pharmaceutically acceptable esters or thioesters include, but are notlimited to, esters or thioesters of the formula --OOCR or --SOCR,wherein R is a pharmaceutically acceptable alkyl, alkenyl, aryl,alkylaryl, arylalkyl, spingosine or substituted spingolipid groups, suchas propionate, enanthate, cypionate, succinate, decanoate andphenylpropionate esters.

Pharmaceutically acceptable ethers or thioethers include, but are notlimited to, ethers or thioethers of the formula --OR or --SR, wherein Ris as defined above or enol, or --OR⁴ is an unsubstituted or substitutedspirooxirane or --SR is a spirothiane.

Suitable sugar residues include, but are not limited to monosaccharides,disaccharides and oligosaccharides, such as a glucuronate.

Pharmaceutically acceptable inorganic esters include, but are notlimited to, inorganic esters of the formula --OSO₂ R⁵ or --OPOR⁵ R⁶,wherein R⁵ and R⁶ are independently --OH, pharmaceutically acceptableesters, pharmaceutically acceptable ethers or pharmaceuticallyacceptable salts.

Other suitable DHEA congeners can be readily identified by administeringthe DHEA congener in the burn model described herein and noting itsanti-progressive ischemic effect.

It is known that reperfusion injury, infarctions and traumatic injury,such as myocardial infarctions, burns, major surgery, chemical burns,blunt trauma, lacerations and the like, can lead to injury in whichtissue necrosis extends beyond the directly affected tissue to includesurrounding unaffected tissue. This ischemia plays an important role indefining the ultimate tissue pathology observed as a consequence oftraumatic injury in humans (10). It is also known that one consequenceof thermal injury is bacterial translo-cation. Thermal injury, i.e.,burns, is the best studied traumatic injury in which progressiveischemia occurs.

The loss of viable skin through the process of progressive ischemicnecrosis contributes significantly to much of the skin loss thatrequires surgical grafting following burn injury (37). A number ofanimal models have been developed which mimic very closely many aspectsof clinical burns. For example, following the administration of anexperimental full-thickness scald burn which covers >20% of the totalbody surface area to rodents (e.g., 72° C. hot water exposure for 7seconds), the immediate tissue effects of the burn injury appear quitemoderate, compared to the extensive damage to the affected andsurrounding skin tissue which develops over the subsequent 24-72 hourperiod. Thus, it has been observed in both clinical and experimentalburns that the total amount of skin lost to a severe thermal injuryrepresents the sum of the immediate direct tissue destruction plus thelatent damage that occurs to the epidermis, dermis and inclusive skinstructures of the affected and surrounding skin areas.

Initial investigations using the dorsal skin thermal injury model inrodents led to some dramatic findings. It was discovered that scaldburn-injured mice that are treated within one hour after thermal injurywith the weakly androgenic steroid hormone, dehydroepiandrosterone(DHEA), develop and resolve their wounds in a manner quite distinct fromuntreated or sham treated thermally injured controls. By 3-4 days afterthermal injury, all control-injured animals demonstrate third and fourthdegree damage to the vast majority of skin tissue within the injurysite. Virtually all of the skin within the affected area is ultimatelylost as a consequence of progressive ischemic necrosis. The extent oftissue damage in these animals associates with a major loss in skinstructures (hair follicles, blood vessels, neurons, and sebaceousglands), an infiltration of fibroblasts, extensive wound contraction,and the formation of numerous fibrous adhesions under the affected skinarea. The DHEA-treated animals (about 2 mg/kg/day after an initialloading dose of 4 mg/kg), however, are observed to develop significantlyless pathology, with much less evidence of progressive damage to thedermis, subdermis and associated skin structures. Whilere-epithelialization is active in both the burn control and theDHEA-treated injured groups of mice, DHEA-treated mice demonstrate muchless wound contraction with notably less formation of fibrous adhesionsunderlying the wound site.

With the use of the dorsal skin injury model, it was clearlydemonstrated that DHEA treatment exerts a very positive influence onwound progression. These findings suggested that treatment of thermallyinjured animals with DHEA may influence wound healing based on afundamental capacity to prevent ischemia. Consequently, a modificationof the procedure first described by Boykin et al., (13) and Eriksson etal., (38) was developed to permit a kinetic evaluation andquantification of progressive dermal ischemia during the immediate andlater phases of thermally-injured mouse ears. The technique employed inthese studies facilitated a rigorous and sequential monitoring of thetime-dependent progression of tissue damage and ischemic necrosis inmouse ears subjected to a hot water scald burn (52° C. for 24 seconds),and has become a valid animal model for investigating progressiveischemia of burn-injured tissue.

The mouse ear consists of two layers of skin, cartilage, sparse musclecells and connective tissue. Organization of the ear vasculature iswell-ordered, comprised of arterioles, precapillary arterioles,post-capillary venules and venules. Employing an apparatus capable ofadministering controlled thermal injury to the entire surface area ofthe mouse ear, researchers have reported observing an immediate changein blood flow patterns. As a result of precise morpho-logical studies onhemodynamic changes following burn injury of the mouse ear, threedistinct zones, easily separable by the degree of pathology, have beendescribed. These zones comprise the zone of complete capillaryocclusion, the zone of partial occlusion (stasis), and the zone ofcapillary hyperemia (13). By one hour after injury, the area of totalcapillary occlusion is restricted to the distal margin of the mouse ear.Located more proximally to this outermost, and immediately sensitivearea, is the zone of partial occlusion or stasis. It is this major areaof ear tissue which becomes progressively ischemic over the 24-72 hourperiod following thermal injury, and which ultimately undergoesnecrosis. Finally, the most proximal area of the affected ear is thezone of hyperemia. This area is fairly resistant to progressivepost-burn ischemia.

It has been discovered that the administration to a patient of atherapeutically effective amount of a DHEA congener in a physiologicallyacceptable carrier as early as possible, preferably within four hours ofa reperfusion injury, infarction or traumatic injury, results in theprevention or the reduction of reperfusion injury, infarction ortraumatic injury-associated ischemia. The prevention or reduction of theischemia results in prevention or reduction of the consequent necrosisof tissue associated with such ischemia. This reduction in ischemiaresults from the reduction of adherence of neutrophils to endothelialcells, as shown in the Example. As a consequence of the reducedneutrophil adherence, the neutrophils do not become activated and do notproduce cellular factors which lead to platelet aggregation. It is mostpreferred that the DHEA congner be administered within two hours of thepatient's sustaining the reperfusion, infarction or traumatic injury.The DHEA congener is administered to patients in ester or otherpharmaceutically acceptable form and within binders, elixirs or otherpharmaceutically acceptable mixtures, or with other pharmaceuticallyacceptable carriers. It is preferred that the DHEA congener beadministered by intravenous injection.

Pharmaceutical compositions containing a compound of the presentinvention as the active ingredient in intimate admixture with apharmaceutical carrier can be prepared according to conventionalpharmaceutical compounding techniques. The carrier may take a widevariety of forms depending on the form of preparation desired foradministration, e.g., intravenous, oral or parenteral. In preparing thecompositions in oral dosage form, any of the usual pharmaceutical mediamay be employed, such as, for example, water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like in thecase of oral liquid preparations (such as, for example, suspensions,elixirs and solutions); or carriers such as starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations (such as, for example,powders, capsules and tablets). If desired, tablets may be sugar-coatedor enteric-coated by standard techniques. For parenterals, the carrierwill usually comprise sterile water, though other ingredients, forexample, to aid solubility or for preservative purposes, may beincluded. Injectable suspensions may also be prepared, in which caseappropriate liquid carders, suspending agents and the like may beemployed.

The dose of the DHEA congener should be based on well knownpharmaceutically acceptable principles, e.g., 1-200 mg/kg, preferably2-50 mg/kg, of the active ingredient. The dose may be administered dailyor every other day, and may be taken as single or multiple doses to givethe proper daily dosage. For unprotected compounds, i.e., those whichcan be sulfated by human sulfotransferases or sulfatases, it ispreferred to administer an excess dose to insure that sufficient activeagent is administered, especially if sulfatases are not active at thesite of tissue injury. It has now been discovered that DHEA-S, ifadministered in a sufficiently high dose such that it is converted tothe dosage of DHEA, can be used to prevent progressive ischemia. It ispreferred that unprotected compounds be administered daily, whereasprotected compounds can be administered either daily or every other day.It is preferred to administer the DHEA congener intrvenously. The dosageof DHEA congener utilized will deliver an equivalent of 10-100 mg/kg ofDHEA. The dose of DHEA-S necessary to deliver this level of DHEA is10-1,000 mg/kg, preferably 50-800 mg/kg. for The patient is treated withthe DHEA congener for 3-30 days, preferably 7-14 days, following theinfarction or traumatic injury.

For those patients who are at high risk for a mycocardial infarction orat risk for reperfusion injury, it is possible to prevent or reduceprogressive ischemia associated with such an infarction or reperfusioninjury by administering the DHEA congener prior to, simultaneouslyand/or following the infarction or reperfusion injury in the dosagesdescribed above. The treatment following the myocardial infarction is asdescribed above. The DHEA congener can be administered to such a patientwho demonstrates the classical signs for an imminent myocardialinfarction in the same manner as described above for treatment followingsuch an infarction.

For those patients who are at risk of bacterial translocation, suchbacterial translocation is prevented or reduced by administering theDHEA congener as described above in the dosages described above. Theadministration to prevent or reduce bacterial translocation continuesuntil the patient is no longer at risk for the bacterial translocation.

It has been discovered that it is critical that the DHEA congener beadministered soon after reperfusion injury, infarction or traumaticinjury in order to prevent or reduce any cellular damage. If theadministration of these compounds occurs too late, blood vessels willbecome occluded (initially with neutrophils adhering to endothelialcells), at which point the administration of these compounds will beunable to prevent or reduce the ischemia. The time frame within whichthe administration should begin may be dependent on the type ofreperfusion injury, infarction or traumatic injury, and can be readilydetermined by appropriate animal models. However, it is preferred thatadministration of the DHEA congener commence within four hours, and mostpreferably within two hours of the ischemia, infarction or traumaticinjury. The administration of the DHEA congeners to prevent or reducebacterial translocation should begin within 24 hours of the injury orstress-causing event. It is preferred that administration of thesecompounds to prevent or reduce bacterial translocation begin within fourhours, and most preferably within two hours. The administration of theDHEA congeners to prevent or reduce ARDS should begin before the onsetof clinical symptoms. Generally, the compounds will be administered topatients at risk of ARDS.

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

EXAMPLE 1 Experimental Thermal-Injury Model

An experimental thermal injury model employing mouse ears was developedwhere temperature and exposure time were established empirically. Theconditions represented the minimal burn injury which progressed to totaltissue necrosis in the exposed ear of untreated mice by 24-72 hourspost-burn. Groups of Balb/c mice, approximately nine weeks old, weregiven an identifying mark, and then divided into control and treatedsubgroups. The thickness of the ear to be immersed in hot water wasrecorded, and then the entire ear of the anesthetized mouse was dippedinto 52° C. water for exactly 24 seconds. Each mouse was returned to itscage after an injection of either the propylene glycol vehicle (control)or 100 mg of test agent dissolved in propylene glycol. Ear swellingchanges were monitored on individual mice at pre-burn, and at varioushours after thermal injury.

EXAMPLE 2 Effect of DHEA in the Thermal-Injury Model

Groups of Balb/c mice, approximately 9 weeks old, were given anidentifying mark, and then divided into control and treated subgroups.The thickness of the ear to be immersed in hot water was recorded, andthen the entire ear of the anesthetized mouse was dipped into 52° C.water for exactly 24 seconds. Each mouse was returned to its cage afteran injection of either the propylene glycol vehicle (control) or 100 mgof DHEA agent dissolved in propylene glycol. Ear swelling changes weremonitored on individual mice at pre-burn, and at 1, 3, 6, 9, 12, 18, 24and 48 hours after thermal injury.

The results of the analysis of edema formation and resolution in theears of control and DHEA-treated mice are shown in FIG. 1. Ear swelling,as a measure of edema, reached a peak in both DHEA-treated and untreatedburned mice by six hours after injury. In the untreated group, theextent of swelling started to decline within 12 hours, and continued todecline rapidly over the subsequent 12 hour periods. Between 24 and 48hours post-burn, ear measurements had to be discontinued in theuntreated group due to the complete loss of ear tissue resulting fromthe complete micro-vascular occlusion of the original zone of stasis.The kinetic analysis of edema in untreated and DHEA-treatedthermally-injured mice showed that the events which take place duringthe first 24 hours following a burn-induced injury are critical to theviability of the thermally-injured tissue, such that the eventualpreservation of viable ear tissue at 48 hours correlates inversely withthe rate at which the swelling response recedes between the peak at sixhours and the final 48 hour time period.

In addition to the analysis of edema in untreated and DHEA-treatedthermally-injured mice, the changes in viability of the ear tissueitself were documented photographically. Injury of the ear tissue inmice given only the vehicle was extensive, with greater than 70% of theear tissue being necrotic and destroyed within 48 hours. The totalaffected area appeared to encompass both The zone of complete vascularocclusion and the original zone of stasis. This latter zone becamedamaged as a secondary consequence of thermal injury, a condition whichdefines progressive post-burn dermal ischemia. However, DHEA-treatedmice showed little injury and the preservation of burned ear tissue wasseen in a kinetic fashion. The only area of ear tissue that was markedlyaffected by, but not lost to the effects of thermal injury correspondedto only the original zone of complete vascular occlusion.

EXAMPLE 3 Effect of Various Compounds in the Thermal Injury Model

Groups of nine-week old thermally injured Balb/c. mice were divided intosubgroups given either vehicle alone, DHEA, androstenediol,16a-bromo-DHEA, andros-tenedione or the potent anti-glucocorticoid,RU486. Individual mice received 100 mg of the indicated steroids or thevehicle alone immediately post-burn (day 0), and further 50 mg dosesevery 24 hours for the duration of the experiment. The ear swellingresponse of each individually marked mouse was recorded at the pre-burnstage, and at 12, 24 and 48 hours post-burn.

Burned ears of mice being treated therapeutically with androstenediol,DHEA, or the non-metabolizable, synthetic derivative of DHEA,16a-bromo-DHEA, each developed significant ear-swelling in response toburn injury (FIG. 2) and exhibited a slow and constant rate ofresolution of the swelling. This slow loss of edema following thermalinjury of the ear was paralleled by only minimal dermal ischemia andnecrosis in the area. The results of this study also confirmed that thedevelopment of edema within the burned ear of untreated mice peaks andthen recedes somewhat rapidly, such that between 24-48 hours post-burn asignificant amount of tissue ischemia and necrosis takes place. Thesimilar pattern of edema followed by progressive ischemic necrosis wasobserved with andostenedione-treated mice. Likewise, a similar patternof edema followed by progressive ischemic necrosis was observed in thegroup of thermally injured animals treated with RU486, indicating thatDHEA is not working solely via its anti-glucocorticoid effects.

FIGS. 3A-3C demonstrate the capacity of DHEA, androstenediol and16α-bromo-DHEA to protect against most of the ischemic consequences ofthermal injury to the ear. Mice treated with either one of these steroidhormones incur early changes in ear tissue with slight to no loss of eartissue several days after thermal injury. The affected area appears tocorrespond to the zone of complete occlusion defined by Boykin (13).Mice given the vehicle alone, androstenedione or RU486 (FIGS. 3D-3F)following thermal injury lose >70% of the exposed ear tissue over thefirst 48 hours post-injury due to progressive post-burn ischemicnecrosis. Without effective treatment, the areas of the burn-injured earwhich became necrotic corresponded to the zone of complete occlusionplus the zone of stasis. Thus, it was demonstrated that treatment ofthermally-injured mice with either DHEA, androstenediol, or16a-bromo-DHEA not only changes the natural course of the edema producedin the ear but also somehow protects the affected tissue fromprogressive damage by inhibit-ing the development of ischemia within thezone of stasis and the ultimate development of necrosis of this area.

In similar experiments, it was found that 16α-hydroxy-DHEA was lessprotective, i.e., reduced the extent of, but did not totally preventprogressive ischemia, and 16α-chloro-DHEA was slightly protectiveagainst progressive ischemia.

EXAMPLE 4 Timing of Initial Administration of DHEA

An experiment was designed to determine whether intervention using DHEAmust be delivered immediately, or whether the intervention can bedelayed for up to several hours following burn injury. Mice wereanesthetized, administered a burn and then, while under anesthesia, fourmice were given vehicle alone, four mice were given 100 mg DHEA, and theremaining mice were divided into additional groups of four. All of themice in a single group would receive 100 mg DHEA either one, two, fouror six hours after thermal injury. Tissue loss by each mouse wasevaluated 72 hours after thermal injury, and the results of the scoringare presented in FIG. 4.

This Figure demonstrates that intervention using DHEA can be delayed forup to two hours with no significant difference in the protective effectsof DHEA mean grade of 1.25% 0.25 (p=<0.001). Even with a delay of fourhours before administration of DHEA, a mean score of 2.75% 0.479 wasobserved (p=<0.016). With a six-hour delay in delivery of DHEA, the meanscore in tissue loss was 4.0% 0.408 and was determined to besignificantly different from the group that received DHEA immediatelyafter thermal injury (p=<0.058). It was concluded that the events whichlead to necrosis are reversible by administration of DHEA for up toseveral hours post-thermal injury.

The above examples demonstrate that moderate-intensity thermal injury ofthe mouse ear is a reliable and reproducible model for examiningprogressive ischemic necrosis of the skin. The results indicate thatimmediate post-burn use of DHEA has a protective effect on thermalinjury-induced dermal ischemia. In addition to DHEA, several othersteroid hormones have been tested for their therapeutic value (see TableI).

                  TABLE 1                                                         ______________________________________                                        Results of Progressive                                                        Steroid Hormone Tested                                                                            Ischemia Analysis                                         (100 mg/mouse)      (mouse ear model)                                         ______________________________________                                        DHEA-S              nonprotective                                             DHEA                protective                                                16a-Bromo-DHEA      protective                                                androstenediol      protective                                                androstenedione     nonprotective                                             RU 486              nonprotective                                             ______________________________________                                    

Along with DHEA, androstenediol and 16a-bromo-DHEA were markedlyprotective, in that 90-100% of the ear tissue remained intact until theexperiment was terminated at two weeks, when the healing process wascomplete. 16a-Hydroxy-DHEA was less protective and 16a-chloro-DHEA wasslightly protective. However, DHEA-sulfate at the dose examined,androstenedione and RU486 were completely nonprotective, in that eardamage and tissue loss equivalent to untreated controls was evident inall animals within 48 hours after thermal injury. It has now beendiscovered that if a sufficiently high dose of DHEA-S is administered tolead to the equivalent amount of DHEA as used in this experiment, thenDHEA-S is protective. The ability to separate protective fromnonprotective steroids in this thermal injury model is most likelyfacilitated by the fact that direct or immediate burn damage to the eartissue is minimal, and that the vast majority of the damage which occursemanates from the progressive ischemia and necrosis caused by the hostresponse to the scald-burn.

The results implicate DHEA, not one of its natural metabolites, as theagent mediating protection against progressive ischemic necrosis. Thebasis for this conclusion is straightforward. 16a-Bromo-DHEA is a DHEAanalog which cannot be effectively metabolized to downstream androgensteroids, and its protective effect is identical to DHEA. Androstenediolalso displays a biologic effect identical to DHEA in the thermal-injuryear model. This steroid is a natural metabolite of DHEA which, throughenzyme-dependent modification, can be converted back to DHEA. It canalso be further metabolized to testosterone. Androstenedione, also asecondary metabolite of DHEA, can be metabolized only to downstreamproducts, (e.g., testosterone and estrogens), with no known conversionto DHEA. Because androstenedione cannot protect against progressivedermal ischemia in this model, its lack of effect supports theconclusion that the active steroid is DHEA, not a downstream androgen orestrogen.

DHEA possesses the published ability to overcome some of the biologicaleffects caused by glucocorticoids. The possibility that DHEA functionsas an anti-glucocorticoid in the dermal ischemia model of thethermally-injured mouse ear was tested by administering the knownanti-glucocorticoid, RU 486, to mice immediately after administration ofthe burn injury. As presented in this model and under the conditionstested, substances with anti-glucocorticoid activities offered nobenefit.

EXAMPLE 5 Effect of DHEA on Reperfusion Injury

Male Sprague-Dawley rats weighing 130-170 g were randomly assigned to nopre-treatment, vehicle pre-treatment or DHEA pre-treatment (4 mg/kg).Animals were treated with vehicle or DHEA the day before and the day ofsurgery. Anesthesia was induced with intraperitoneal pentobarbital(60-70 mg/kg). The rats were placed on a heating pad, and bodytemperature (measured by rectal probe) was maintained at between 35°-37°C. Detection of the cremaster muscle on its neurovascular pedicle wasperformed according to conventional techniques (39-41). Briefly, a skinincision is made from the anterior iliac spine to the tip of thescrotum. The testis with cremaster muscle intact is then dissected awayfrom the scrotum. An opening of 1 cm is made on the ventral surface ofthe cremaster, and the testis and spermatic cord are removed. Under amicroscope, the neurovascular pedicle, consisting of thepubic-epigastric arteries, vein, and genitofemoral nerve, is thencompletely isolated by dissecting to the origin of the vessels from theexternal iliac artery and vein. Finally, the front wall of the cremastermuscle sac is opened and the island cremaster muscle flap is preparedfor intravital videomicroscopy. The rat is secured on a speciallydesigned tissue bath, and the cremaster muscle flap is spread over thecoverglass in the opening at the bottom of the bath and fixed with 5-0silk sutures. It is then transilluminated from below, using a fiberoptictungsten lamp. The muscle is kept moist and covered with impermeableplastic film. The tissue bath, designed specifically for temperaturecontrol, is filled with 0.9% saline and the temperature maintained atbetween 35° C.-36° C. The microscope is equipped with a color videocamera. The video image oft he microcirculation is displayed on a 19"monitor, where the final magnification is x 1800. Measurement ofmicrovascular activity is recorded after isolation of the muscle toestablish the pre-ischemia baseline. After proper positioning of clampsto completely shut down blood flow to the muscle flap, the duration ofthe ischemic period is six hours. Following removal of clamps to inducereperfusion injury, activity in the microvasculature is measured at 30,60 and 90 minutes post-reperfusion. In all experimental subjects,ischemia is followed by reflow and then by an initial period of flow ofblood through the microcirculation. This burst of circulatory activityis followed by marked reperfusion injury that induces loss of flow.

The following parameters are used to evaluate the state of the cremastermuscle microvasculatory system prior to ischemia and after reperfusion.

1) Density of Perfused Capillaries. The density of perfused capillariesin each of three flap regions (Zone 1, 2 and 3) is measured by countingthe number of flowing capillaries in proximity to the preselectedpostcapillary venule. Nine visual fields of capillaries are counted ateach postcapillary venule site, for a total of 27 fields per cremastermuscle flap. Results are shown in FIGS. 5A, 5B and 5C for Zones 1, 2 and3, respectively.

2) Leukocyte Count in Postcapillary Venules. Video scans of threepre-selected postcapillary venules are taken in proximal, middle anddistal flap regions. For each venule, the number of leukocytes rollingthrough the luman, the number adhering to the endothelium and the numberhaving migrated across the endothelium over a two-minute period arerecorded. Results are shown in FIGS. 6A, 6B and 6C for rollers, strikersand diopedesis, respectively.

3) Red Blood Cell Velocities in A1 (First Order) and A2 (Second Order)Arterioles. Red blood cell velo-cities are recorded in the mainarterioles of the cremaster flap using a custom-made optical Dopplervelocimeter. Results are shown in FIGS. 7A and 7B, for velocity ofvenous and arterial blood, respectively.

A. Reperfusion Injury in Untreated and Vehicle-Treated Rats

Six rats were untreated and six rats were pre-treated with vehicle.Under conditions of six hours of ischemia and 90 minutes of reperfusion,the absolute number of rolling, sticking and transmigrated leuko-cytesincreased dramatically within 60 minutes of reperfusion and showed afurther increase at 90 minutes (FIGS. 6A-6C). A dramatic decrease wasobserved in the absolute number of perfused capillaries per high-poweredfield that were at both 30 and 60 minutes post-reperfusion, with acontinued decrease in numbers of flowing capillaries at 90 minutespost-reperfusion (FIGS. 5A-5C). Likewise, red cell velocities inA2-sized vessels were significantly slower at 60 and 90 minutespost-reperfusion (FIGS. 7A and 7B).

B. Reperfusion Injury in DHEA-Treated Rats

Under conditions where rats were pre-treated with 4 mg/kg DHEA bysubcutaneous injection the day before and the day of surgery, a markedand highly significant protective effect of the therapy was measured.All three parameters exhibited values that were close to, or identicalwith normal values. Of major importance, it was noted that alltimepoints, endothelial-adherent properties were unchanged from baselinevalues. This conclusion is based on the fact that numbers of roll-ing,sticking and transmigrating leukocytes appeared remarkably similar tobaseline values (FIGS. 6A-6C). Red cell velocities in A2 arterioles wereslower to return to normal rates of flow, with velocities in some areasmeasuring 75% of normal at 90 minutes post-reperfusion (FIGS. 7A and7B). At the 90-minute timepoint, the number of capillaries flowing inthe microvasculature were not significantly different from the baselinevalues obtained prior to ischemia (FIGS. 5A-5C).

Without being bound by any theory of the physiolo-gical and biochemicaloperation of the DHEA congeners, it is believed that the anti-ischemiceffects of these compounds are due to their activity on the adhesion ofneutrophils to endothelial cells. Thus, these compounds are effective inpreventing or reducing ischemia which may result from other types oftissue injury, which can be modulated by affecting adhesion toendothelial cells. This inhibition of neutrophil adhesion preventsactivation of neutrophils and transmigration to the tissue side of theendothelium. Since transmigration of neutrophils is inhibited,neutrophil-induced massive damage to endothelial cells and parenchymalcells is prevented. Since neutrophil activation is prevented, productionof cellular factors Coy neutrophils) which leads to platelet aggregationis also prevented. Thus, progressive tissue necrosis is prevented orreduced. In addition, the progressive ischemia of gut tissue (leading tobacterial translocation) and of the epidermis and of cardiac muscle andthe ischemia of the alveolar wall (leading to ARDS) are mediated throughsimilar mechanisms. Thus, these compounds are also effective inpreventing or reducing bacterial translocation and ARDS.

EXAMPLE 6 Effect of DHEA on Expression of P-Selectin by Platelets

Platelets were fractionated from freshly dram blood (mature adults andelderly). Platelets were either utilized unwashed or washed. Washedplatelets were obtained by conventional procedures (42, 43). Briefly,blood was collected to a syringe containing 1 volume of anticoagulant(0.085M sodium citrate, 0.065M citric acid, 2% dextrose) to 7 volumes ofblood. Routinely, 50 ml of blood was withdrawn, Blood samples werecentrifuged at 180×g fro 15 minutes at room temperature to sediment redand white blood cells. The upper two-thirds of the platelet-rich plasmasupernatant was carefully removed by aspiration, and the platelets werepelleted by centrifugation at 1100×g for 10 minutes at room temperature.The supernatant was decanted and the platelets were resuspended bygently mixing the sample in 2 ml of washing buffer (Tyrode's bufferwithout calicium, pH 6.50 at 37° C.). The platelet suspensioin was thendiluted to a volume equal to the original volume of blood drawn withTyrode's buffer, and centrifuged at 1100×g for 10 minutes at roomtemperature. The platelets were washed twice more by centrifugation andresuspended in 5 ml of incubaiton buffer (washing buffer adujsted to pH7.4 at 37° C.). The platelets were counted in a Neubauer hemocytometer.

Washed and unwashed platelets were examined for the presence ofP-selectin by direct immunostaining. Platelets (1×10⁶) were incubatedwith phycoerythrin-conjugated either negative control antibody oranti-human P-selectin monoclonal antibody (CD62 antibody, CAMFolio,Becton-Dickinson) for 15 minutes on ice. After that time, samples werewashed twice with staining buffer (PBS, 0.1% sodium azide, 2% fetalbovine serum), reconstitued in 500 μl of staining buffer and analyzed bya FACS can flow cytometer (Becton Dickinson). The fluorescence wasdsiplayed as a single parameter histogram on a linear scale.

Measurement of P-selectin levels on surface of washed platelets obtainedfrom blood of mature individuals showed that approximatley 50% of washedplatelets (resting platelets) tested positive for the presence ofP-selectin. Sixty-eight percent of the unwashed platelets obtained fromblood of an elderly individual tested positive for P-selectin. Whenwhole blood form this individual was supplemented with 10 μM finalconcentraion of DHEA prior to fractionation of the platelets and thentest, only 12% of the platelets stained positive for P-selectin. Thisdown-regulation of P-selectin by DHEA was accompanied by a 40% reductionin thrombin activated platelet aggregation. When this latter individualwas placed on a supplemental therapy with DHEA-S and the plateletsfractioned from blood drawn during the supplemental therapy with DHEA-S,the platelets were refractory to exogenous DHEA when activated with thesame amount of thrombin as activated prior to the therapy. Thus, theobserved down-regulation of P-selectin on the surface of platelets fromelderly individuals by DHEA was accompanied by a prevention ofthrombin-stimualted aggregation of these platelets by DHEA.

EXAMPLE 7 Effect of DHEA on Expression of P-Selectin by EndothelialCells

Non-virally transformed Human Dermal Microsvascular Endothelial cellswere cultured using conventional techniques. Cells in passage number 2were put on cover slips covered with attachment factor, and were grownin serum free system without phebol red until they became confluent.Groups of cells were incubated with vehicle alone or with 1 μM, 10 μM,25 μM, 50 μM or 100 μM DHEA at 37° C. for 10 minutes. The cells werethen activated with 10⁻⁵ M histamine or with Dulbecco's phosphate buffersaline (dPBS) at 37° C. for 5 minutes.

The cells were then examined by indirect immunostaining/fluorescencemicroscopy. Briefly the cells were first washed 2-3 times in dPBScontaing 1% bovine serum albumin (BSA), 1-2 minutes per wash. The cellswere then fixed in ice-cold methanol for 5-7 minutes and then washed 2-3times in dPBS containing 1% BSA and 0.01% azide. The cells were thenincubated with anti P-selectin antibody at 4° C. in a humified chamberfor 30 minutes. The cells were then washed 2-3 times in dPBS containing1% BSA at 4° C., 1-2 minutes per wash. The cells were then incubated ananti anti-body linked to P-phycoerytherin at 4° C. for 30-40 minutes,after which the cells were washed 2-3 times in dPBS containing 1% BSA at4° C., 1-2 minutes per wash. The slides are then mounted and andP-selectin expression on endothelium is examined in fluorescencemicroscopy using rhodamine filterset.

Similary results are noted as seen for P-selectin expression inplatelets. Namely, DHEA at concentrations of 10 μM or greater preventedthe up-regulation of P-selectin expression normally observed onendothelium in response to histamine. The endothelium incubated withDHEA prior to histamine activation looked similar to the control,non-activated endothelium.

It will be appreciated that the methods and com-positions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

LIST OF REFERENCES

(1) Rodgers, G. M. (1988). FASEB J 2:116-123.

(2) Hernandez, L. A. et al. (1987). Am. J. Physiol. 253 (Heart Cir.Physiol. 22):H699-H703.

(3) Lucchesi, B. R. (1990). Am. J. Cardiology 65:14I-23I.

(4) Lehr, H. A. et al. (199). J. Clin. Invest. 87:2036-2041.

(5) Entman, M. L. et al. (1991). FASEB J 5:2529-2537.

(6) Weyrich, A. S. et al. (1993). J. Clin. Invest. 91:2620-2629.

(7) Lefer, A. M. et al. (1991). FASEB J 5:2029-2034.

(8) Brown, J. M. et al. (1988). J. Clin. Invest. 81:1297-1301.

(9) "Cellular Injury and Adaptation," in Pathologic Basis of Disease,Cotran et al., eds., WB Saunders, Philadelphia, pp. 1-81 (1989).

(10) Robson, M. C. et al. (1979). Plastic and Recons-tructive Surgery63:781-787.

(11) Robson, M. C. et al. (1980). J. Trauma 20:722-725

(12) Rockwell, W. B. and Ehrlich, H. P. (1992). J. Burn Care Rehab13:403-406.

(13) Boykin, J. V. et al. (1980). Plastic Reconstruct. Surgery.66:191-198.

(14) Erhlich, H. P. (1984). J. Trauma 24:311-318.

(15) Erhlich, H. P. (1987). J. Trauma 27:420-424.

(16) Mileski, W. et al. (1992). J. Sugg. Res. 52:334-339.

(17) Morehouse, J. L. et al. (1986). Gastroenterol 91:673-682.

(18) Maejimak, et al. (1984). Arch. Surg. 119:166-172.

(19) Czaja, A. J. et al. (1974). N. Engl. J. Med. 291:925-929

(20) Seavitt, S. (1967). Br. J. Surg. 54:32-41.

(21) Desai, M. H. et al. (1991). Surgery Gyn. Obstet, 172:257-261.

(22) Deitch, E. A. and R. Berg (1987). J. Burn Rehab. 8:475-482.

(23) Edmiston, C. E. and R. E. Condon (1991). Surgery, Gyn. Obstet.173:73-83.

(24) Deitch, E. A. (1990). Arch. Surg. 125:403-404.

(25) Saadia, R. et al. (1990). Br. J. Surg. 77:487-492.

(26) Mainous, M. R. et al. (1991). Arch. Surg. 126:33-7.

(27) Vaughan, W. G. et al. (1992). J. Ped. Surg. 27:968-973.

(28) Deitch, E. A. et al (1992). Circ. Shock 36:206-16.

(29) Fukushima, R. et al. (1992). Ann. Surg. 216:438-444.

(30) Tokyay, R. et al. (1992). J. Trauma 32:704-713.

(31) Simon, R. H. and Ward, P. A. (1992). In Inflamma-tion: BasicPrinciples and Clinical Correlates, 2d Ed., Galin, J. I. et al., Eds.,Raven Press, Ltd., New York, pp. 999-1016.

(32) Araneo, B. A. et al. (1993). Arch. Surg. 128:318-325.

(33) Eich, D. M. et al. (1992). U.S. Pat. No. 5,110,810.

(34) Eich, D. M. et al. (1992). U.S. Pat. No. 5,162,198.

(35) Nestler, J. E. et al. (1990). U.S. Pat. No. 4,920,115.

(36) Kent, S. (1982). Geriatrics 37:157-159.

(37) Jackson (1953). British J. Surg. 40:588-593.

(38) Ericksson, E. et al. (1980). Microvascular Res. 19:374-379.

(39) Anderson, G. L. et al. (1988). Microvascular Res. 36:56-63.

(40) Siemionow, M. et al. (1991 ). Microcirc. Endoth. Lymphatics2:183-197.

(41) Siemionow, M. et al. (1993). J. Hand Surgery. 18A:963-971.

(42) Orlinska, U., (1989). PhD Dissertation: Transforming growth factorβ1 and plyamines in monocrotaline-induced pulmonary hypertension. Univ.of Kentucky, School of Pharmacy, Lexington, Ky.

(43) Hawrylowicz, C. H. et al. (1989). J. Immunol. 143:4015-4018.

What is claimed is:
 1. A method for preventing or reducing loss oftissue viability caused by adhesion of neutrophils to endothelial cells,which comprises administering to a patient having a tissue injurysimultaneously with or within six hours following said tissue injury, atherapeutically effective amount of a compound of the formula ##STR2##wherein X is H or halogen;R¹, R² and R³ are independently ═O, --OH,--SH, H, halogen, pharmaceutically acceptable ester, pharmaceuticallyacceptable thioester, pharmaceutically acceptable ether,pharmaceutically acceptable thioether, pharmaceutically acceptableinorganic esters, pharmaceutically acceptable monosaccharide,disaccharide or oligosaccharide, spirooxirane, spirothirane, --OS O₂ R⁵or --OPOR⁵ R⁶ ; R⁵ and R⁶ are independently --OH, pharmaceuticallyacceptable esters or pharmaceutically acceptable ethers; andpharmaceutically acceptable salts,with the proviso that when (a) R³ is Hor OH, R² is ═O or OH and X is H or (b) R³ is H or OH, R² is ═O and X ishalogen, then R¹ is not OR⁴, wherein R⁴ is H, fatty acid, C₁₋₁₀ alkyl,C₁₋₁₀ alkenyl, C₁₋₁₀ acetylenic, (Y)_(n) -phenyl-C₁₋₅ -alkyl, (Y)_(n)-phenyl-C₁₋₅ -alkenyl or --CO--R⁷, R⁷ is H, fatty acid, C₁₋₁₀ alkyl,C₁₋₁₀ alkenyl, C₁₋₁₀ acetylenic, (Y)_(n) -phenyl-C₁₋₅ -alkyl, (Y)_(n)-phenyl-C₁₋₅ -alkenyl, Y is the same or different and is halogen, C₁₋₄alkyl, C₁₋₄ alkenyl, alkoxy, carboxy, nitro, sulfate, sulfonyl, C₁₋₄carboxylesters or C₁₋₄ thioesters, and n is 0, 1, 2 or
 3. 2. The methodof claim 1, wherein said tissue injury is a reperfusion injury of anyvascularized tissue.
 3. The method of claim 2, wherein said compound isadministered prior to the tissue injury.
 4. The method of claim 3wherein said compound is administered intravenously.
 5. The method ofclaim 1, wherein said patient has a traumatic injury, and the traumaticinjury is a result of thermal injury, surgery, chemical burns, blunttrauma or lacerations.
 6. The method of claim 5, wherein the traumaticinjury is thermal injury.
 7. The method of claim 1, wherein said patienthas an infarction.
 8. The method of claim 7, wherein the infarction is amyocardial infarction.
 9. The method of claim 1, wherein said compoundis administered within four hours of the tissue injury.
 10. The methodof claim 9 wherein said compound is administered intravenously.
 11. Themethod of claim 1, wherein said compound is administered within twohours of the tissue injury.
 12. The method of claim 11 wherein saidcompound is administered intravenously.
 13. The method of claim 1,wherein the compound is administered in the amount of 1 to 200 mg/kg.14. The method of claim 13, wherein the compound is administered in theamount of 2 to 50 mg/kg.
 15. The method of claim 1, wherein the compoundis administered in single or multiple doses.
 16. The method of claim 1,wherein R³ is OH.
 17. The method of claim 1 wherein said compound isadministered intravenously.